A mosaic genetic screen identifies GlcT as a tumor suppressor in adult Drosophila midgut

(A) A diagram showing lineage hierarchy of intestinal stem cells (ISCs) in the Drosophila intestine. Abbreviations: EB, enteroblast; EEP, enteroendocrine cell; EC, enterocyte; EE, enteroendocrine cell. (B-D) Compared to control clones (green), the EA30 and E230 clones induced at day 5 are larger and contain a higher number of Pros+ cells within each clone. GlcT null allele (GlcTΔ8) mutant clones exhibit a similar phenotype. (E-F) Expression of UAS-GlcT in EA30 or E230 mutant clones rescues the tumor phenotype. (G-J) Depletion of GlcT in progenitor cells using RNAi with esg-GAL4ts leads to ISC proliferation (G-H, stained with Phospho-Histone 3) and excessive EEC generation in the midgut (I-J). For G-J, flies were shifted to 29°C for 14 days. Error bars represent the mean ± SEM, with p-values indicated in the figure (two-tailed Student’s t-test). Scale bars: 25 μm.

Genetic analysis of the components of the GSL synthesis pathway reveals specific tumor-suppressive activity for egh, in addition to GlcT

(A) A diagram illustrating a portion of the metabolic pathways of glycosphingolipids (GSLs) synthesized from ceramide, highlighting the key enzymes involved at each step. (B) GlcTΔ8 mutant clones without or with co-expression of p35 or CDase stained with anti-DCP-1 or anti-Pros, as indicated. Clones were examined at day 7-10 after clone induction. (C-D) Quantitative analysis of the percentage of Pros+ cells in clones of specified genotypes. (E) Genetic analysis of the key enzymes in the GSL synthesis pathway. Note that egh mutant clones (eghA, eghB, or egh7) exhibited a phenotype of increased EEs similar to that observed in GlcT mutant clones, but Brn228, β4GalNAcTA-IR, and α4GT1-IR clones did not. (F, G) Quantitative analysis of cell number (F) and the percentage of Pros+ cells (G) in clones of specified genotypes. For E-G, clones were examined at day 7 after clone induction. Error bars represent the mean ± SEM, with p-values indicated in the figure (two-tailed Student’s t-test). Scale bars: 25 μm.

The EE tumor phenotype is a result of MacCer deficiency

(A) A diagram showing the major compartments along the length of the Drosophila midgut, including the anterior (yellow), middle (Region 3, R3, green), and posterior midgut (blue). (B-C) Feeding GlcT mutant flies with LacCer alleviated the overgrowth and excessive EE phenotype, which was observed in clones of both the anterior and posterior midgut. Note that the suppressive effect was more pronounced in the anterior midgut compared to the posterior midgut. Error bars represent the mean ± SEM, with p-values indicated in the figure (two-tailed Student’s t-test). Scale bar: 25 μm.

Loss of GlcT leads to reduced activation of Notch signaling in ISC progenies

(A-B) The expression of NRE-lacZ, the Notch activity reporter, in GlcT mutant clones. Note that the fluorescence intensity of LacZ was significantly reduced within GlcT mutant clones compared to the cells outside of the clone. (C) GlcT mutant clones in the posterior (R5) region of the Drosophila midgut stained with anti-Tk (left) or anti-AstC (right). The two EE subtypes at R5 region are normally present in a 1:1 ratio. Note that AstC+ EEs, but not TK+ EEs, were present in GlcT mutant clones. (D) The effect of forced activation of Notch in GlcT mutant clones by expressing Nintra. Error bars represent the mean ± SEM, with p-values indicated in the figure (two-tailed Student’s t-test). Scale bars: 25 μm.

GlcT regulates the endocytic trafficking of Delta

(A-B) Anti-Pros staining in flies carrying GlcT-IR driven by Dltsand NREts respectively. Flies were shifted to 29°C for 14 days before analysis. (C) In a 3-hour Dl antibody uptake assay performed in the Drosophila midgut, Dl in ISCs of GlcT mutant clones (green box) showed reduced membrane localization and increased intracellular fluorescence compared to that in ISCs of control clones (see the enlarged inset boxes). (D, F) ISCs in esgts>GlcT-RNAi flies exhibited increased accumulation of Dl in early endosomes (Rab5+, with co-localization indicated by yellow arrows). (E-F) The percentage of Dl+ late endosomes (Rab7+) was largely unchanged. For D-F, flies were shifted to 29°C for 10 days before analysis. Co-localization analysis was performed using the intensity-based quantification feature in Leica Application Suite X (LAS X) software, with the Overlap Coefficient (ranging from 0 to 1) employed to quantify co-localization (1 indicating perfect co-localization and 0 indicating no co-localization). Error bars represent the mean ± SEM, with p-values indicated in the figure (two-tailed Student’s t-test). Scale bar: 25 μm, unless otherwise noted.

GlcT shows tissue specificity in regulating Notch signaling activity

(A) The expression of Cut in a GlcT mutant clone (green area) in wing disc spanning the dorsal-ventral boundary. (B) The expression of EYA in GlcT mutant follicle cell clones (non-green areas). Note that there was no obvious difference in the level of EYA expression inside and outside of the clones. (C) Punctate accumulation of Dl was observed in GlcT mutant germ cell clones (non-green area, indicated by arrows in the enlarged inset), but not in the germline of a wild-type developing egg chamber (see enlarged inset with the dashed yellow line). Scale bars: 25 μm.

Transient loss of UGCG in mouse small intestine causes reduced number of ISCs and increased number of goblet cells

(A-B) The expression of the Notch target gene Olfm4 was downregulated in villin-CreERT2, UGCGflox/flox mice 48 hours post-induction (n=3). The CKO mice displayed shorter villi (above the dotted line) and elongated crypts (below the dotted line). (C-D) The number of Muc2+ goblet cells was significantly increased in the CKO mice. (E) A schematic model for the role of MacCer in regulating the Notch signaling pathway. In signaling-sending cells, mutations in GlcT result in the loss of the downstream metabolite MacCer, leading to a rapid endocytosis of the membrane Dl and an accumulation of Dl in early endosomes. This causes a reduced level of Notch signaling activation in signaling-receiving cells. Error bars represent the mean ± SEM, with p-values indicated in the figure (two-tailed Student’s t-test). Scale bars: 100 μm.